Application method of clonidine in treatment of noise-induced hearing loss

Abstract

The invention belongs to the technical field of new application of medicines, discloses a new application method of clonidine in treatment of noise-induced hearing loss, and aims to improve hearing loss caused by noise. Clonidine is given before noise, the hearing threshold after noise is detected through ABR, and it is found that compared with a noise group without drug administration, the ABR threshold of a mouse given with the noise group of clonidine is obviously improved on the first day (PE1) after noise. Meanwhile, ABR thresholds on the seventh day (PE7) and the fourteenth day (PE14) after noise are tracked, and the hearing threshold of a mouse in a noise group given with clonidine is still obviously lower than that of a mouse in a noise group without drug administration. And inner ear tissue pathological damage caused by noise-induced hearing loss is relieved. A confocal microscope observes that PE1, PE7 and PE14 of mouse outer hair cells of a noise group which are not administered are obviously lost, zonal synapses are obviously reduced, and the mouse outer hair cells and zonal synapses of a colon noise group are not obviously changed.

Description

Technical Field

[0001] The present invention belongs to the technical field of new uses of drugs, and particularly relates to a new application method of clonidine in the treatment of noise-induced hearing loss. Background Art

[0002] Noise-induced hearing loss (NIHL) is the most common occupational disease, and 12% or more of the global population is at risk of deafness caused by noise. Prolonged or repeated exposure to noise environments above 85 dB can lead to significant hearing loss. The acute hearing loss in NIHL includes temporary threshold shift (TTS) that can recover to normal hearing thresholds and permanent threshold shift (PTS) that cannot recover. Among them, PTS is the main cause of hearing disability. Therefore, the drug prevention and treatment of noise-induced deafness have always been the focus and difficulty in otological research. Currently, there is still a lack of effective drugs for preventing and treating noise-induced hearing loss.

[0003] Clonidine is a drug approved by the US Food and Drug Administration (FDA) with multiple clinical uses. It exerts its pharmacological effects mainly by stimulating the brainstem α2-adrenergic receptors, thereby reducing the activity of the sympathetic nervous system and the release of norepinephrine. Currently, studies have found that the elevated serum norepinephrine concentration in animal models after noise exposure can lead to impaired hearing function. Therefore, it is predictable that clonidine can protect against noise-induced hearing loss by reducing the serum norepinephrine concentration.

[0004] In the prior art, clonidine is mainly used to treat moderate and severe hypertension, hypertension in patients with glaucoma, and is also used for migraine, severe dysmenorrhea, menopausal hot flashes, and glaucoma.

[0005] Through the above analysis, the problems and defects of the prior art are as follows:

[0006] Currently, there is still a lack of effective drugs for preventing and treating noise-induced hearing loss. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a new application method of clonidine in the treatment of noise-induced hearing loss.

[0008] The present invention is implemented as follows. A new application method of clonidine in the treatment of noise-induced hearing loss includes:

[0009] Step 1, subject mice administered with clonidine and non-administered mice to noise exposure for 6 hours using 110 dB SPL white noise, and inject clonidine before the noise exposure;

[0010] Step 2, observe the treatment effects at 0 hour, 12 hours, 24 hours, 72 hours, 7 days, and 14 days after the noise exposure;

[0011] This includes statistically analyzing the hearing conditions of the three groups of mice through ABR audiometry, observing the number of sensory hair cells and ribbon synapses under a confocal microscope with immunofluorescence staining of hair cell spreads, and observing the protein expression levels of adrenergic receptor α2A, calcium channel TRPC6, etc. through immunofluorescence staining and Western blot experiments;

[0012] Step 3, evaluate the roles of the above proteins in noise exposure by separately knocking out adrenergic receptor α2A and calcium channel protein TRPC6.

[0013] Furthermore, the administration method is: intraperitoneal injection.

[0014] Furthermore, the administration dose is: 1 mg / kg.

[0015] Furthermore, the administration schedule is: administer the drug 24 hours before the noise exposure, 1 hour before the noise exposure, and immediately after the noise exposure.

[0016] Combined with the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solution to be protected by the present invention from the following aspects:

[0017] First, regarding the technical problems existing in the above prior art and the difficulty of solving such problems, closely combining the technical solution to be protected by the present invention and the results and data during the R & D process, analyze in detail and profoundly how the technical solution of the present invention solves the technical problems and the creative technical effects brought about after solving the problems. The specific description is as follows:

[0018] The purpose of the present invention is to provide a new use of clonidine for the treatment of noise-induced hearing loss, and by injecting clonidine, the symptoms of noise-induced deafness can be alleviated and the hearing of patients can be improved.

[0019] 1. Improve the hearing loss caused by noise. Clonidine was administered before the noise exposure, and the hearing thresholds after the noise exposure were detected by ABR. The present invention found that compared with the noise group without drug administration, the ABR thresholds of the mice in the noise group administered with clonidine were significantly improved on the first day after the noise exposure (PE1). At the same time, the ABR thresholds on the 7th day (PE7) and the 14th day (PE14) after the noise exposure were tracked, and the hearing thresholds of the mice in the noise group administered with clonidine were still significantly lower than those of the mice in the noise group without drug administration.

[0020] 2. Alleviate the pathological damage of the inner ear tissue caused by noise-induced hearing loss. It was observed under a confocal microscope that the outer hair cells of the mice in the noise group without drug administration were significantly lost at PE1, PE7, and PE14, and the ribbon synapses were significantly reduced, while there were no obvious changes in the outer hair cells and ribbon synapses of the mice in the noise group administered with clonidine.

[0021] Clonidine is a drug with multiple clinical uses approved by the US Food and Drug Administration (FDA) and is currently mature in clinical applications. The present invention proposes a new use of clonidine in the treatment of noise-induced hearing loss. Clonidine can reduce the hearing threshold after noise exposure and maintain the integrity of hair cells and ribbon synapses in the cochlea.

[0022] Second, as the creative auxiliary evidence of the claims of the present invention, it is also reflected in the following important aspects:

[0023] (1) The expected benefits and commercial value after the transformation of the technical solution of the present invention are:

[0024] As the first solution to expand the application of the marketed drug clonidine to the treatment of noise-induced hearing loss, its transformation potential is significant. More than 12% of the global population is at risk of noise-induced deafness, and the groups involved in high-noise exposure industries such as manufacturing, military, and aviation are large. The traditional treatment solutions mainly focus on physical protection, while this solution provides the feasibility of drug intervention by verifying the protective effects of clonidine on cochlear hair cells and synapses. Since clonidine is a safe drug approved by the FDA, the clinical transformation cycle and cost are greatly reduced. After commercialization, it can be developed into an occupational health protection drug, or cooperate with hearing aid manufacturers to build a combined solution of "drug protection + device treatment" to cover the two scenarios of prevention and treatment, with a market potential of tens of billions of dollars.

[0025] (2) The technical solution of the present invention fills the technical gaps in the domestic and international industries:

[0026] In the prior art, the research on drug treatment for noise-induced deafness at home and abroad has long focused on traditional directions such as antioxidants and neurotrophic factors, but no clinical breakthroughs have been achieved. The present invention first reveals the key connection between adrenergic system regulation and noise-induced hearing loss, creatively discovers the reversible protection mechanism of α2A receptor agonists, and establishes a protein regulation model of the TRPC6 calcium channel. Through the dual technical paths of multi-time point dynamic monitoring (0h - 14 days) and gene knockout verification, for the first time internationally, the protection map of clonidine on the hair cell-synapse axis is drawn, filling the knowledge gap and clinical practice vacancy in the field of targeted drug treatment for noise-induced deafness.

[0027] (3) Whether the technical solution of the present invention solves the technical problems that people have always been eager to solve but have never succeeded in:

[0028] Noise-induced permanent threshold shift (PTS), as the main cause of occupational hearing disability, has long troubled the medical community with its irreversible damage mechanism. Traditional treatments can only delay the disease process and cannot repair damaged hair cells and synaptic connections. This invention breakthroughly regulates the concentration of norepinephrine, and in an animal model, it maintains the basic hearing threshold at the PE14 (14 days after exposure) stage, especially extending the dosing window to after noise exposure (the "immediate dosing after noise" protocol), which is of milestone significance for the clinical treatment of sudden noise-induced injuries (such as explosion shock). Experimental data show that the ribbon synapse retention rate in the drug-treated group exceeds 80%, overcoming the internationally recognized technical bottleneck of synaptic regeneration. Brief Description of the Drawings

[0029] Figure 1 It is a flowchart of a new application method of clonidine in the treatment of noise-induced hearing loss provided by an embodiment of the present invention.

[0030] Figure 2 It is a process flowchart of a new application method of clonidine in the treatment of noise-induced hearing loss provided by an embodiment of the present invention.

[0031] Figure 3 It is the use of clonidine provided by an embodiment of the present invention to attenuate noise-induced hearing loss in C57BL / 6J mice. (A) ABR thresholds and threshold shifts of three groups of mice at PE1 (1 day after noise exposure), PE7 (7 days after noise exposure), and PE14 (14 days after noise exposure). The CON group shows the baseline ABR hearing function. (CON: non-intervention control group; NE: noise exposure group; NE + CLO: noise exposure plus clonidine intervention group) Data are expressed as mean ± standard deviation. *P < 0.05, P < 0.01, *P < 0.001, comparing the NE group and the NE + CLO group, n = 15 in each group.

[0032] Figure 4 It is that clonidine provided by an embodiment of the present invention reduces noise-induced cochlear base turn hair cell loss. (A) Confocal images show the change in the number of cochlear hair cells from the apex to the base at PE1, PE7, and PE14. Hair cells are labeled with Myo7a (red), cell frameworks are labeled with phalloidin (green), and cell nuclei are labeled with DAPI (blue). Asterisks indicate OHC loss. (B - D) The number of OHCs per 100 μm cochlear basilar membrane length. Scale bar = 20 μm. *P < 0.001, P < 0.0001, comparing the NE group and the NE + CLO group, n = 3 in each group.

[0033] Figure 5Clonidine reduces noise-induced cochlear ribbon synapse loss provided by the embodiments of the present invention. (A) By co-localization of Ctbp2 (red) and GluA2 (green), the number of cochlear ribbon synapses at PE1 was compared among three groups of mice after noise exposure. (B) Quantification of ribbon synapses under each inner hair cell. Scale bar = 10 μm. *P<0.05, P<0.01, comparing the NE group and the NE+CLO group, n = 3 in each group. Detailed implementation manners

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] As Figure 1 、 Figure 2 shown, a new application method of clonidine in the treatment of noise-induced hearing loss provided by the embodiments of the present invention includes the following steps:

[0036] S101, subject mice administered with clonidine and non-administered mice to noise exposure for 6 hours using 110 dB SPL white noise, and inject clonidine before the noise.

[0037] S102, observe the treatment effect at 0 hour, 12 hours, 24 hours, 72 hours, 7 days, and 14 days after the noise.

[0038] It includes statistically analyzing the hearing of three groups of mice by ABR audiometry, observing the number of sensory hair cells and ribbon synapses under a confocal microscope through hair cell spreading immunofluorescence, and observing the protein expression of adrenergic receptor α2A, calcium channel TRPC6, etc. through immunofluorescence staining and Western blot experiments.

[0039] S103, evaluate the role of the above proteins in noise exposure by respectively knocking out adrenergic receptor α2A and calcium channel protein TRPC6.

[0040] The administration method provided by the embodiments of the present invention: intraperitoneal injection.

[0041] The administration dose provided by the embodiments of the present invention: 1 mg / kg.

[0042] The administration regimen provided by the embodiments of the present invention: 24 h before the noise, 1 hour before the noise, and immediately after the noise.

[0043] First, further refine the observation in the time dimension. Set more grouped sampling points after noise exposure (such as 0 hours, 12 hours, 24 hours, 72 hours, etc.), and use advanced imaging and quantitative histological analysis to synchronously monitor the dynamic changes of inner ear hair cells, supporting cells and stria vascularis structures, so as to accurately characterize the differential mechanisms of transient and delayed hearing loss.

[0044] Second, combine gene and drug dual intervention strategies. In addition to the conventional clonidine injection, introduce molecular targeting drugs that inhibit specific pathways (such as oxidative stress or apoptosis pathways), and use RNA interference or gene editing means to knockdown key proteins, so as to evaluate the synergistic effect of specific signaling pathways in hearing protection and improve the intervention accuracy and treatment prospects of this experiment for noise-induced deafness.

[0045] To exert the timeliness of clonidine, the present invention selects three different drug administration time points: 24 hours before noise exposure, 1 hour before, and immediately after noise exposure by intraperitoneal injection. The purpose of this drug administration strategy is to reduce the long-term stress response of mice during and after noise exposure and control the elevated norepinephrine level caused thereby.

[0046] The experimental results show that on the first day after noise exposure (PE1), the auditory brainstem response (ABR) thresholds of the experimental group treated with clonidine (NE + CLO group) at three test frequencies of 8 kHz, 16 kHz and 32 kHz are significantly lower than those of the control group only exposed to noise (NE group) (see Figure 3 ). Especially at the frequency of 32 kHz, the hearing threshold of the NE + CLO group is 10 dB lower than that of the NE group, and this difference is statistically significant. In addition, even on the seventh day (PE7) and fourteenth day (PE14) after noise exposure, the hearing thresholds of the NE + CLO group in the high-frequency range are still significantly lower than those of the NE group, indicating that clonidine has a lasting protective effect on the high-frequency hearing threshold shift caused by NIHL. These findings reveal the potential utility of clonidine in the prevention and treatment of NIHL, especially in reducing high-frequency hearing loss.

[0047] The present invention uses Myo7a (myosin label, red), phalloidin (cytoskeleton label, green) and DPAI (nucleus label, blue) immunostaining techniques, combined with a confocal microscope, to quantitatively analyze the number of outer hair cells (OHC) and inner hair cells (IHC) in the apical, middle and basal turns of the cochlear basilar membrane. This method aims to evaluate the degree of hair cell damage at different time points (PE1, PE7 and PE14) after noise exposure.

[0048] The results showed that compared with the NE group exposed only to noise, the number of OHC losses in the basal turn region of the NE + CLO group treated with clonidine was significantly reduced. In addition, in the apical and middle turn regions, the degree of OHC loss in the NE + CLO group was relatively smaller (see Figure 4 ). Since the basal turn hair cells are mainly responsible for high-frequency hearing responses, the relatively fewer hair cell losses in the basal turn region of the NE + CLO group corresponded to the improvement in ABR hearing thresholds at 32 kHz. This finding further confirmed the positive effect of clonidine in attenuating noise-induced hearing loss.

[0049] The data of the present invention indicate that clonidine treatment can effectively protect the basal turn OHCs from noise damage, which is crucial for maintaining high-frequency hearing. In addition, the relative preservation of OHCs in the apical and middle turn regions suggests that clonidine has a general protective effect on hair cells in different regions of the cochlea.

[0050] In addition to the assessment of the number of hair cell losses, the present invention also performed a detailed analysis of the number and morphology of ribbon synapses in the apical, middle, and basal turn regions of the cochlear basilar membrane of mice in each group on the first day after noise exposure (PE1). Using Ctbp2 (marking the presynaptic membrane, in red) and GluA2 (marking the postsynaptic membrane, in green) immunostaining methods and combined with confocal microscopy technology, the present invention quantitatively evaluated the loss of ribbon synapses.

[0051] The research results showed that compared with the NE group exposed only to noise, the number of ribbon synapses in the apical and middle turn regions of the NE + CLO group treated with clonidine was significantly higher than that of the NE group (see Figure 5 ). This finding suggests that clonidine can not only reduce the reduction in the number of ribbon synapses caused by noise exposure, but also maintain the morphological integrity of synapses, manifested by fewer cases of misalignment between the presynaptic membrane and the postsynaptic membrane in the NE + CLO group, that is, there is a better synaptic morphology.

[0052] It is worth noting that the damage of ribbon synapses is related to the occurrence of temporary threshold shift (TTS). The results of ribbon synapse counting in the present invention are consistent with the previous hearing test results at PE1, further confirming the protective effect of clonidine in attenuating TTS caused by noise. However, in the basal turn region, no obvious improvement was observed in the number and morphology of ribbon synapses. This phenomenon may be related to the greater impact of noise on the high-frequency region and the basal turn. On the one hand, clonidine may not be sufficient to fully maintain the improvement effect on the basal turn ribbon synapses; on the other hand, due to the loss of hair cells in the basal turn region, the corresponding synaptic structures are also lost.

[0053] Example 1: Experimental study on the preventive treatment effect of clonidine on noise-induced hearing loss

[0054] Sixty healthy C57BL / 6J mice were randomly divided into three groups: a control group, a noise exposure group, and a clonidine intervention group, with 20 mice in each group. All mice were conventionally raised before the experiment, and their basic auditory thresholds were measured. The mice in the clonidine intervention group were given clonidine by intraperitoneal injection 24 hours and 1 hour before noise exposure and immediately after noise exposure, at a dose of 1 mg / kg; the control group and the noise exposure group were injected with an equal volume of normal saline.

[0055] All mice were exposed to white noise at 110 dB SPL for 6 hours to simulate noise-induced hearing loss. After noise exposure, auditory brainstem response (ABR) tests were performed at 0 hours, 12 hours, 24 hours, 72 hours, 7 days, and 14 days to measure the changes in auditory thresholds of the mice at different frequencies.

[0056] The results showed that the auditory thresholds of the noise exposure group were significantly increased at each time point after noise exposure, indicating hearing loss. The auditory thresholds of the clonidine intervention group at the same time points were significantly lower than those of the noise exposure group, indicating that clonidine has a significant protective effect on noise-induced hearing loss.

[0057] Furthermore, through immunofluorescence staining and confocal microscopy observation, it was found that the number of sensory hair cells and ribbon synapses in the mice of the clonidine intervention group was significantly higher than that of the noise exposure group after noise exposure, indicating that clonidine has a protective effect on sensory hair cells and synaptic structures.

[0058] In addition, Western blot experiments showed that the expression levels of adrenergic receptor α2A and calcium channel TRPC6 in the mice of the clonidine intervention group were significantly lower than those of the noise exposure group, indicating that clonidine reduced noise-induced auditory damage by inhibiting the activation of related receptors and ion channels.

[0059] In summary, administering clonidine before and immediately after noise exposure can effectively reduce auditory function damage and has a significant preventive effect on noise-induced hearing loss.

[0060] Example 2: Therapeutic effect experiment of clonidine on noise-induced hearing loss

[0061] Forty C57BL / 6J mice were randomly divided into a noise exposure group and a clonidine treatment group, with 20 mice in each group. All mice were conventionally raised before the experiment, and their basic auditory thresholds were recorded. All mice were exposed to white noise at 110 dB SPL for 6 hours to induce noise-induced hearing loss.

[0062] After noise exposure was completed, the mice in the clonidine treatment group were immediately given intraperitoneal injection at a dose of 1 mg / kg, and the administration was repeated at 24 hours and 72 hours; the mice in the noise exposure group were injected with an equal volume of normal saline at the same time points.

[0063] ABR tests were conducted at 0 hours, 12 hours, 24 hours, 72 hours, 7 days, and 14 days after noise exposure to measure the recovery of auditory thresholds. The experimental results showed that the recovery rate of auditory thresholds in the clonidine treatment group was significantly faster than that in the noise exposure group at each time point, especially at 7 days and 14 days, showing a more significant recovery effect.

[0064] Immunofluorescence staining and Western blotting experiments showed that in the mice of the clonidine treatment group, the damage degree of hair cells and ribbon synapses was significantly reduced after noise exposure, and the expression levels of adrenergic receptor α2A and calcium channel TRPC6 were significantly decreased compared with those in the noise exposure group.

[0065] At the same time, through comparative experiments on mice with knockout of adrenergic receptor α2A and TRPC6 genes, it was found that the protective effect of clonidine was significantly weakened in the mice with the above gene knockout, indicating that its therapeutic effect on hearing loss is closely related to the expression of these two proteins.

[0066] In summary, timely treatment with clonidine after noise exposure can significantly promote the recovery of auditory function and reduce the damage of sensory hair cells and ribbon synapses, showing a significant therapeutic effect.

[0067] The above description is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. A pharmaceutical composition for the treatment of noise-induced hearing loss, characterized in that, The pharmaceutical composition comprises an effective dose of clonidine and a pharmaceutically acceptable carrier or excipient; wherein the clonidine is administered by intraperitoneal injection, with a single-dose of 1 mg / kg, and is administered respectively 24 hours before noise exposure, 1 hour before noise exposure, and immediately after noise exposure, so as to improve or restore the auditory function impairment caused by noise exposure.

2. The pharmaceutical composition for treating noise-induced hearing loss according to claim 1, wherein, The pharmaceutical composition inhibits the activities of adrenergic receptor α2A and calcium channel TRPC6, thereby reducing the damage of sensory hair cells and ribbon synapses caused by noise exposure, improving auditory sensitivity and promoting the recovery of auditory function.

3. A new application method of clonidine in the treatment of noise-induced hearing loss, characterized in that, The new application method of clonidine in the treatment of noise-induced hearing loss comprises the following steps: Step 1, exposing mice administered with clonidine and non-administered mice to 110 dB SPL white noise for 6 hours, with clonidine injected before the noise. Step 2, observing the treatment effects at 0 hour, 12 hours, 24 hours, 72 hours, 7 days, and 14 days after the noise. Including counting the hearing conditions of the three groups of mice by ABR audiometry, observing the number of sensory hair cells and ribbon synapses under a confocal microscope with immunofluorescent staining of hair cell spreads, and observing the protein expression of adrenergic receptor α2A, calcium channel TRPC6, etc. through immunofluorescent staining and Western blot experiments. Step 3, evaluating the roles of the above proteins in noise exposure by respectively knocking out adrenergic receptor α2A and calcium channel protein TRPC6.

4. The new application method of clonidine in the treatment of noise-induced hearing loss according to claim 3, characterized in that, The administration method: intraperitoneal injection.

5. The new application method of clonidine in the treatment of noise-induced hearing loss according to claim 3, characterized in that, The administration dose: 1 mg / kg.

6. The new application method of clonidine in the treatment of noise-induced hearing loss according to claim 3, characterized in that, The administration schedule: administered 24 hours before the noise, 1 hour before the noise, and immediately after the noise.

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